CD34-positive acute promyelocytic leukemia is associated with leukocytosis, microgranular/hypogranular morphology, expression of CD2 and bcr3 isoform.

Acute promyelocytic leukemia (APL) has a favorable prognosis. Current therapy includes chemotherapy used in combination with all-trans-retinoic acid (ATRA). Although the differentiating effects of ATRA on promyelocytes have been well established, in vitro studies have shown that less-differentiated APL blasts (CD34(+)) demonstrate a variable responsiveness to ATRA. To assess the clinical relevance of this finding, we analyzed a cohort of 38 patients with t(15;17) and/or PML-RARalpha APL to determine the incidence and laboratory features of CD34(+) APL. Thirty-two percent (12/38) of cases were CD34(+). There was a difference in WBC at presentation between CD34(+) and CD34(-) cases (34.6 +/- 9.2, mean +/- standard error vs. 5.4 +/- 2.0, P = 0.009). Patients with CD34(+) APL demonstrated a micro/hypogranular phenotype (75%) (P = 0.001), co-expression of CD2(+) (83%) (P = 0.001), and the bcr3 isoform (100%) (P = 0.017). In contrast, CD34(-) cases demonstrated hypergranular morphology (65%), CD2(+) (15%), and the bcr1 isoform (50%). A high presenting WBC count (\G10 x 10(9)/L) was associated with an inferior overall survival (Log rank = 0.0047). Patients with CD34(+) APL demonstrated an incidence of early mortality of 50%. Despite a marked correlation between CD34 positivity and increased WBC count, overall survival of CD34(+) and CD34(-) cases did not differ significantly in our small cohort. Immunophenotypic analysis for CD34 expression should be included in future large APL trials to determine if detection of CD34(+) blasts represents an independent adverse prognostic factor.

Morphology of acute promyelocytic leukemia with cytogenetic or molecular evidence for the diagnosis: characterization of additional microgranular variants.

Early diagnosis of t(15;17) acute promyelocytic leukemia (APL) is essential because of the associated disseminated intravascular coagulation and the unique response of the disease to all-trans retinoic acid (ATRA) therapy. Early diagnosis depends primarily on morphological recognition. The French-American-British (FAB) classification, however, does not describe all morphological variations that occur in APL. In 25 cases with evidence of APL confirmed by cytogenetic and/or molecular analysis, we found a heterogeneous morphological group. The most common form of APL was heterogeneous and consisted of various combinations of cells in which hypergranular cells and some cells with multiple Auer rods were obvious. In some cases, one cell predominated. This led to the description of five subcategories. These included the classical FAB M3 with hypergranular cells and multiple Auer rods; the FAB variant with hypogranular bilobed cells; the basophilic cell type of McKenna et al. [Br. J. Haematol 50:201, 1982]; and two additional subtypes, one consisting of differentiated promyelocytes and a few blast cells (M2-like), and the other consisting largely of blast cells and a few early promyelocytes (M1-like). Immunophenotyping revealed a pattern of CD33 and/or CD13 positivity, and CD14 and HLA-DR negativity in 96% of cases. CD2 was positive in the FAB variant and in the subtype with basophilic cells, but negative with other subtypes. Three out of five cases with basophilic cell predominance [McKenna et al.: Br J Haematol 50:201, 1982], and one out of two M2-like cases, responded to ATRA therapy. Awareness of the heterogeneity and the atypical morphologic subtypes found in t(15;17) APL will contribute to improved recognition and early institution of ATRA therapy.

Monoclonal antibodies in the management of acute leukemia.

This report reviews the diagnostic significance of immune markers, their relationship to patient outcome, and the therapeutic uses of monoclonal antibodies (MoAbs) in acute leukemia. Immunophenotyping allows for rapid and reproducible diagnosis in the majority of cases of acute leukemia. It is of particular importance in recognizing the major immunologic subclasses of acute lymphoblastic leukemia (ALL), and in identifying subtypes of acute myeloblastic leukemia (AML) which cannot be differentiated by morphology and cytochemistry alone, such as FAB M0 or M7. Immune marker analysis has been used to detect minimal residual disease in patients' bone marrow and CSF after treatment. However, the presence of leukemia-associated phenotypes on small numbers of normal cells may reduce the sensitivity of detection in some cases. The prognostic value of immune markers in AML is limited. In ALL, the prognostic significance of the different immunophenotypic subtypes has been lessened by modern treatment protocols. The relationship of mixed-lineage or biphenotypic antigen expression to patient outcome in both AML and ALL is unclear. Therapeutic applications of MoAbs in acute leukemia include immunologic techniques for purging malignant cells from autografts prior to transplantation, T-lymphocyte depletion from allografts as a strategy to reduce graft-versus-host disease, and the use of flow cytometry to monitor the timing and extent of leukapheresis in peripheral stem cell transplantation. MoAbs have also enabled the recent development of transplantation protocols using positively-selected CD34+ stem cells.

CD2+, CD3-, CD56/NCAM+ malignant lymphoma with TCR beta gene rearrangement: a case report.

A case of CD56/NCAM+ malignant lymphoma is reported. Only a rare malignant lymphoma cell showed azurophilic granules in the cytoplasm of Giesma-stained preparations, while electron microscopic examination revealed occasional cytoplasmic granules with paracrystalline inclusions. The most common phenotype seen in NK lymphomas, CD2+, CD3-, CD56+, CD16-, CD57-, was present in the case. Cases with this phenotype have been interpreted to represent either true NK lymphoma or T-cell lymphoma with NK expression. Genotyping, where performed, has shown TCR germline configuration. Our case showed TCR beta rearrangement indicating that the above phenotype can be associated with a peripheral T-cell lymphoma.

Prolonged hemorrhagic shock does not impair regeneration of plasma coagulant masses in the rabbit.

BACKGROUND AND METHODS: Twelve adult male albino rabbits were assigned alternately to normotensive and hypotensive groups to assess the effect of hypovolemic shock on spontaneous correction of dilutional coagulopathy. All animals underwent dilutional exchange transfusion with 200 mL of rabbit RBCs and 5% human albumin. Half the animals were then acutely hemorrhaged and subsequent aliquots of blood removed as needed to maintain the mean arterial pressure at 40 mm Hg. RESULTS: By 6 hr after production of dilutional coagulopathy, masses and plasma concentrations of fibrinogen and Factor II had increased modestly but significantly, and Factor VII mass and concentration and in vitro coagulation had returned almost to normal; plasma volume was unchanged in the normotensive animals. In the hypovolemic shock animals, where coagulant mass regeneration was as rapid as in the normotensive animals, a doubling of total plasma volume (p less than .01) prevented the concentrations of fibrinogen and Factor II, and hence the coagulation times, from improving. CONCLUSIONS: Dilutional coagulopathy corrects spontaneously within hours. Normovolemic shock prolongs dilutional coagulopathy not by impairment of factor regeneration but because of further (internal) dilution due to plasma expansion. Rapid correction of dilutional coagulopathy is likely to necessitate cryoprecipitate administration.

A case of CD3+, CD4-, CD8-, WT31- acute T-cell leukemia.

The article reports a case of acute T-cell leukemia (T-ALL) with the unusual CD3+, WT31 phenotype. On surface marker analysis, the blast cells were found to be CD7+, CD2-, CD5-, CD1-, CD4-, CD8-, CD3+ and negative for B-lymphoid and myeloid lineage. The cells had both TCR beta and TCR gamma gene rearrangement but had negative results with the monoclonal antibody WT31, which reacts with a framework epitope on the T alpha/beta heterodimer (Ti) of the conventional T-cell receptor (TCR). This suggests an association of the CD3 molecular complex with a different polypeptide (the alternate TCR gamma). In two similar reported cases of T-ALL (WT31-, CD3+, CD4-, CD8-), the CD3 molecular complex was found to be associated with a product of the T gamma gene.

Distribution of a hematopoietic-specific differentiation antigen of K562 cells in the human myeloid and lymphoid cell lineages.

BALB/c mice were immunized with uninduced K562 erythroleukemia cells and hybridomas were isolated after fusion of immune spleen cells to P3/NS1 murine myeloma cells. One selected hybrid, designated 10L-30, secreted an antibody of subclass immunoglobulin G2a which was specific for hematopoietic cells. Analysis of 10L-30 binding by complement-mediated cytotoxicity, indirect immunofluorescence, solid-phase radioimmunoassay, and mixed hemadsorption assay indicated that the 10L-30 antigen was expressed on the myeloid cell lines K562, KG-1A, KG-1, some B- and T-lymphoid cell lines, and all normal human peripheral blood T-lymphocyte samples tested, but was absent on the more differentiated myeloid cell lines HL-60, ML-2, ML-3, and normal blood granulocytes. Induction of erythroid differentiation in hemin-treated K562 cells caused a 10-fold reduction in 10L-30 binding. Human erythroid and granulocytic progenitor cells, platelets, erythrocytes, and reticulocytes were nonreactive, as were a variety of nonhematopoietic human tumor cell lines. Freshly isolated leukemic bone marrow samples from patients with M5 (2 of 5), M6 (2 of 2), acute lymphoid leukemia (9 of 14), and chronic myeloid leukemia in lymphoid blast crisis (1 of 1) were 10L-30 positive. The combined evidence indicates that the 10L-30 antigen is a normal, hematopoietic-specific differentiation antigen which is strongly expressed on both immature cells of the myeloid lineage and more generally in lymphoid ontogeny. The 10L-30 antigen may be a useful marker of both normal and leukemic hematopoietic differentiation.

Classifying acute leukemia by immunophenotyping: a combined FAB-immunologic classification of AML.

A panel of commercially available monoclonal antibodies and five heteroantisera were used to distinguish and subtype 138 cases of acute leukemia (AL). The immunophenotype was compared with the French-American-British (FAB) classification obtained on the cases. The immunophenotype discriminated acute myelogenous leukemia (AML) from acute lymphoblastic leukemia (ALL) and recognized cases not distinguished by cytochemistry (22% of cases), mixed lineage phenotypes (13% of cases), and cases with separate populations of lymphoblasts and myeloblasts (one case). Using the immunologic panel and derived criteria to subtype AML, correspondence of the immunophenotype to the FAB subtypes M1, M2, M4, and M5 was possible in greater than 80% of cases. A combined classification of the immunophenotype and FAB morphology/cytochemistry was devised for AML subtyping. It is recommended that immunophenotyping should be done at least in all cases with negative or inconclusive cytochemistry. At present, we suggest that until a "gold standard" for identifying leukemic subtypes is developed, the best method for typing acute leukemia is by using a combination of morphology, cytochemistry and immunophenotyping.

The contribution of cytochemistry and immunophenotyping to the reproducibility of the FAB classification in acute leukemia.

Intraobserver and interobserver reproducibility of the FAB classification was assessed for two independent observers whose decisions are acted on for treatment of patients with acute leukemia in the Hamilton region. Intraobserver reproducibility was assessed for Wright-stained preparations that were examined independently on two consecutive occasions at least 2 weeks apart. A third reading was performed with Wright stain and cytochemical data, and the fourth reading was done with addition of immunophenotype data. Concordance was calculated using a statistic that corrects for chance-expected agreement (k), and a weighted statistic that takes into account the seriousness of disagreements was used. Samples were available for morphological and cytochemical assessment on 105 patients, and immunophenotype data were available on 93 specimens. Intraobserver concordance was 64.8% and 70.5% for observers A and B, respectively, with kappa values of .56 and .62. There were 37 discordant readings for observer A and 31 for observer B, with each observer discordant between lymphocytic:nonlymphocytic phenotypes in ten cases. Concordance between observers was 63% (k = .54) and 72% (k = .65) for each of two separate readings for Wright-stained preparations only. Reproducibility improved to 89% (k = .86) when cytochemistry was added. When immunophenotype information was provided in addition to Wright-stained and cytochemical preparations, the agreement was 99%. Lymphocytic:nonlymphocytic discordance between observers occurred on nine occasions when Wright-stained preparations only were available and four times when cytochemistry was added; it did not occur with immunophenotyping. The study suggests that immunophenotyping, when added to morphological assessment of acute leukemia, may contribute substantially to agreement between observers.

Platelet fragments do not contribute to elevated levels of platelet associated IgG.

Most assays that measure platelet associated IgG (PAIgG) relate the IgG associated with the test platelets to the platelet count. This could lead to a systematic error if platelet fragments were present in the washed platelet sample but not counted. To address this issue, we studied platelets from patients with idiopathic thrombocytopenic purpura (ITP), thrombocytopenia complicating cardiopulmonary bypass, and laboratory synthesized platelet fragments (freeze-thawed) using electron microscopy. Scanning electron microscopic (SEM) examination of the platelet samples demonstrated appreciable numbers of fragments only in the freeze-thawed specimens. Yet, 'fragments' could be seen in all specimens using transmission electron microscopy (TEM). Most of these 'fragments' proved to be artefacts: we found that the ratio of 'fragments' to intact platelets observed in the TEM specimens was similar to the estimated ratio of 'fragments' to platelets that would have been generated had the specimens been sectioned at 90 degrees to the plane of the actual section. Platelets labelled with the membrane label 125I-iodosulfanilic acid were fragmented by repetitive freeze-thawing. Measurement of pellet radioactivity following washing indicated that the fragments were lost during the washing procedure. These studies indicate that elevated levels of platelet associated IgG in ITP do not represent artefacts due to contamination of the test platelets by platelet fragments.

Coagulopathy related to dilution and hypotension during massive transfusion.

A retrospective review of 64 patients receiving more than 10 units of red cell concentrate plus crystalloid within 12 h revealed two consecutive patterns of elevation of the partial thromboplastin time (PTT). The PTT at 3 to 4 h (PTT3-4) correlated with the number of liters of crystalloid (LC) infused over the first 3 h (PTT3-4 = 37 + 7 LC, r = .7643, p less than .001); the PTT thereafter (PTT4+) correlated with the number of hours of closely antecedent hypotension (AH) (PTT4+ = 37 + 21AH, r = .8680, p less than .001). These data indicate a transient dilutional coagulopathy, followed by coagulopathy related to the duration of closely AH. Whether this latter is due to impaired production, disseminated intravascular coagulation, or dilution due to internal shifts of fluids and/or proteins, remains to be clarified. Therapeutic implications of these data are discussed.

A platelet function defect in preeclampsia.

A prospective study was performed on 26 preeclamptic patients and 17 pregnant control subjects relating the platelet count to in vivo platelet function as assessed by the bleeding time and in vitro platelet function as assessed by collagen-stimulated thromboxane B2 biosynthesis. The results of these tests were normal in all control subjects. Nine of the 26 preeclamptic patients (34%) showed thrombocytopenia, and five of these patients had a prolonged bleeding time. Four of the 16 nonthrombocytopenic patients also had a prolonged bleeding time. Eleven patients had impaired thromboxane B2 biosynthesis, and seven of these had a prolonged bleeding time. In all patients, the bleeding time returned to normal, and in most the platelet count returned to normal within five days or after delivery. A significant proportion of patients with preeclampsia develop an acquired defect of platelet function that could contribute to bleeding.

Simultaneous or sequential expression of lymphoid and myeloid phenotypes in acute leukemia.

Acute mixed myeloid-lymphoid leukemia is uncommon. We report four cases in which myeloid and lymphoid cell markers were observed simultaneously or sequentially when 94 patients with acute leukemia were phenotyped according to the French-American-British (FAB) classification system, with cytochemical stains, and with immunologically defined differentiation markers (identified by monoclonal antibodies and antiterminal deoxynucleotidyl transferase [TdT]). In one case, conversion from acute lymphoblastic leukemia to acute myeloid leukemia was noted (FAB L1, TdT+ to FAB M4, Auer rods, TdT-). In another patient, two distinct populations of myeloid and lymphoid blast cells were observed simultaneously (TdT-, LeuM1+/TdT+, LeuM1-). In two additional patients, acute leukemia was characterized by the expression of both lymphoid and myeloid markers on the same cell (TdT+/Leu M1+, B4+/Leu M1+ and greater than or equal to 70% TdT+, T11+, My9+). The Philadelphia (Ph1) chromosome was negative in all cases, though other chromosomal abnormalities were noted in three out of four cases. Malignant transformation of a pluripotential stem cell for both lymphoid and myeloid lineages, with or without the Ph1 chromosome marker, could explain the coexistence of distinct populations of lymphoblasts and myeloblasts in acute leukemia. Acute leukemia with a biphenotypic profile may reflect genome depression accompanying neoplasia.